α-2- macroglobulin therapies and drug screening methods for Alzheimer's disease.

Abstract

The disclosed invention relates to the finding that the A2M-2 deletion mutation, which is a predisposing factor for Alzheimer's Disease, leads to the production of altered alpha2M RNA transcripts and proteins. Based on this finding, the invention provides for new therapeutic agents for AD, including molecules having Abeta and low density lipoprotein receptor-related protein (LRP) binding domains, peptides, nucleic acid molecules, antisense oligonucleotides, and viral vectors for gene therapy. In addition, the invention relates to pharmaceutical compositions containing these therapeutic agents, methods of using these therapeutic agents to combat Alzheimer's Disease, and methods of screening for therapeutic agents that can combat Alzheimer's Disease.

What is claimed is:  
     
       1. A method of screening for an agent that increases the ratio of A2M-1 mRNA to A2M-2 mRNA comprising: 
       (a) incubating one or more test cells in the presence of a test agent, wherein said test cells are heterozygous or homozygous for the A2M-2 allele; and  
       (b) comparing the ratio of A2M-1 mRNA to A2M-2 mRNA in said test cells to the ratio of A2M-1 mRNA to A2M-2 mRNA in one or more control cells, wherein said control cells are untreated with said test agent and said control cells carry the same number of A2M-2 alleles as said test cells; and wherein a ratio of A2M-1 mRNA to A2M-2 mRNA in said test cells significantly higher than the ratio of A2M-1 mRNA to A2M-2 mRNA in said control cells indicates that said test agent is effective.  
     
     
       2. The method of  claim 1 , wherein a ratio of A2M-1 mRNA to A2M-2 mRNA in said test cells twice said ratio of A2M-1 mRNA to A2M-2 mRNA in said control cells indicates that said test agent is effective. 
     
     
       3. The method of  claim 1 , wherein said test cells are heterozygous for the A2M-2 allele. 
     
     
       4. The method of  claim 1 , wherein said test cells are homozygous for the A2M-2 allele. 
     
     
       5. The method of  claim 1 , wherein said test cells and said control cells are selected from the group consisting of glioma cells, hepatoma cells and hepatocytes. 
     
     
       6. The method of  claim 1 , wherein comparing the ratio of A2M-1 mRNA to A2M-2 mRNA in said test cells to the ratio of A2M-1 mRNA to A2M-2 mRNA in one or more control cells comprises: 
       (a) hybridizing mRNA from said test cells to a probe comprising a polynucleotide sequence complementary to mRNA encoding exon 18 of A2M-1;  
       (b) incubating said mRNA from said test cells with S1 nuclease to produce a first digestion product, wherein said first digestion product comprises A2M mRNA fragments hybridized to said probe, and wherein said A2M mRNA fragments are A2M-1 mRNA fragments and/or A2M-2 mRNA fragments;  
       (c) hybridizing MRNA from said control cells to said probe;  
       (d) incubating said mRNA from said control cells with S1 nuclease to produce a second digestion product, wherein said second digestion product comprises A2M mRNA fragments hybridized to said probe, and wherein said A2M mRNA fragments are A2M-1 mRNA fragments and A2M-2 mRNA fragments;  
       (e) separating said A2M mRNA fragments in said first digestion product according to size and separating said A2M mRNA fragments in said second digestion product according to size, wherein said A2M-1 mRNA fragments are larger than said A2M-2 mRNA fragments; and  
       (f) quantitating the amount of said A2M-1 mRNA fragments and the amount of said A2M-2 mRNA fragments in said first digestion product and the amount of said A2M-1 mRNA fragments and the amount of said A2M-2 mRNA fragments in said second digestion product;  
       (g) comparing the ratio of A2M-1 mRNA to A2M-2 mRNA in said first digestion product to the ratio of A2M-1 mRNA to A2M-2 mRNA in said second digestion product; wherein a ratio of A2M-1 mRNA to A2M-2 mRNA in said first digestion product significantly higher than said ratio of A2M-1 mRNA to A2M-2 mRNA in said second digestion product indicates that said test agent is effective.  
     
     
       7. The method of  claim 6 , wherein said probe is 250-500 nucleotides in length. 
     
     
       8. The method of  claim 7 , wherein said probe comprises a polynucleotide sequence complementary to mRNA encoding exons 17 and 18 of A2M. 
     
     
       9. The method of  claim 7 , wherein said probe comprises a polynucleotide sequence complementary to mRNA encoding exons 17, 18, and 19 of A2M. 
     
     
       10. The method of  claim 9 , wherein said test cells are heterozygous for the A2M-2 allele. 
     
     
       11. The method of  claim 9 , wherein said test cells are homozygous for the A2M-2 allele. 
     
     
       12. The method of  claim 6 , wherein said probe has a sequence complementary to nucleotides 2057-2284 of SEQ ID NO:1. 
     
     
       13. The method of  claim 6 , wherein said probe has a sequence complementary to nucleotides 2024-2323 of SEQ ID NO:1. 
     
     
       14. The method of  claim 6 , wherein said probe has a sequence complementary to nucleotides 2057-2384 of SEQ ID NO:1. 
     
     
       15. The method of  claim 1 , wherein comparing the ratio of A2M-1 mRNA to A2M-2 mRNA in said test cells to the ratio of A2M-1 mRNA to A2M-2 mRNA in one or more control cells comprises: 
       (a) amplifying the mRNA from said test cells using reverse transcriptase polymerase chain reaction (RT-PCR) to produce a first amplification product comprising A2M DNA fragments, wherein a first primer and a second primer are used in said RT-PCR, wherein said first primer and said second primer, when used in PCR to amplify cDNA encoding A2M-1, amplify a region of said cDNA comprising exon 18, and wherein said A2M DNA fragments are A2M-1 DNA fragments and A2M-2 DNA fragments;  
       (b) amplifying the mRNA from said control cells using RT-PCR to produce a second amplification product comprising A2M DNA fragments, wherein said first primer and said second primer are used in said RT-PCR, and wherein said A2M DNA fragments are A2M-1 DNA fragments and/or A2M-2 DNA fragments;  
       (c) separating said A2M DNA fragments in said first amplification product according to size and separating said A2M DNA fragments in said second amplification product according to size, wherein A2M-1 DNA fragments are larger than A2M-2 DNA fragments;  
       (d) quantitating the amount of A2M-1 DNA fragments and the amount of A2M-2 DNA fragments in said first digestion product and the amount of A2M-1 DNA fragments and the amount of A2M-2 DNA fragments in said second digestion product; and  
       (e) comparing the ratio of A2M-1 DNA fragments to A2M-2 DNA fragments in said first amplification product to the ratio of A2M-1 DNA fragments to A2M-2 DNA fragments in said second amplification product; wherein a ratio of A2M-1 DNA fragments to A2M-2 DNA fragments in said first amplification product significantly higher than said ratio of A2M-1 DNA fragments to A2M-2 DNA fragments in said second amplification product indicates that said test agent is effective.  
     
     
       16. The method of  claim 15 , wherein said region comprises exons 17 and 18 of A2M. 
     
     
       17. The method of  claim 15 , wherein said region comprises exons 17, 18, and 19 of A2M. 
     
     
       18. The method of  claim 17 , wherein said test cells are heterozygous for the A2M-2 allele. 
     
     
       19. The method of  claim 17 , wherein said test cells are homozygous for the A2M-2 allele. 
     
     
       20. The method of  claim 17 , wherein said first primer is 8-50 nucleotides in length and said second primer is 8-50 nucleotides in length. 
     
     
       21. The method of  claim 15 , wherein said region has the sequence of nucleotides 2052-2289 of SEQ ID NO:1. 
     
     
       22. The method of  claim 15 , wherein said region has the sequence of nucleotides 2052-2289 of SEQ ID NO:1. 
     
     
       23. The method of  claim 15 , wherein said first primer has a nucleotide sequence complementary to nucleotides 2024-2038 of SEQ ID NO:1, and said second primer has a nucleotide sequence of nucleotides 2309-2323 of SEQ ID NO:1. 
     
     
       24. The method of  claim 15 , wherein said first primer has a nucleotide sequence of nucleotides 2024-2038 of SEQ ID NO:1, and said second primer has a nucleotide sequence complementary to nucleotides 2309-2323 of SEQ ID NO:1.